BD Diagnostic Systems (publ.). Difco Manual (Manual of Microbiological Culture)

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196 The Difco Manual

P. aeruginosa ATCC

27853

E. coli ATCC

25922

Y. ruckeri ATCC

29908

S. aureus ATCC

25923

S. cerevisiae ATCC

9763

B. subtilis ATCC

6633

Fish Peptone No. 1 Section II

User Quality Control

Identity Specifications

Dehydrated Appearance: Beige, free-flowing, homogeneous.

Solution: 1% solution is light to medium amber,

clear to very slightly opalescent, may

have a slight precipitate.

Reaction of 1%

Solution at 25°C: pH 6.7 ± 0.2

Cultural Response

Prepare a 1% concentration of Fish Peptone No. 1 with the

addition of 0.5% sodium chloride. Inoculate test organisms and

incubate for 18-48 hours at 35 ± 2°C. Incubate Vibrio tubiashii

for 18-48 hours at 25 ± 2°C. Saccharomyces cerevisiae is

tested with the addition of 0.5% dextrose. Vibrio tubiashii is

tested with the addition of 1.5% sodium chloride.

INOCULUM

ORGANISM ATCC

CFU GROWTH

Bacillus subtilis† 6633 100-1,000 fair to good

Escherichia coli 25922* 100-1,000 good

Saccharomyces cerevisiae 9763 100-1,000 good

Staphylococcus aureus 25923* 100-1,000 good

Vibrio tubiashii 19105 100-1,000 good

*These cultures are available as Bactrol

Disks and should be

used as directed in Bactrol Disks Technical Information.

†Bacillus subtilis is available as Bacto Subtilis Spore Suspension.

Amino Acids (%)

Alanine 3.48 Lysine 2.51

Arginine 2.19 Methionine 0.83

Aspartic Acid 3.21 Phenylalanine 0.95

Cystine 0.24 Proline 2.19

Glutamic Acid 5.27 Serine 1.27

Glycine 5.29 Threonine 1.17

Histidine 1.54 Tryptophan 0.15

Isoleucine 0.92 Tyrosine 0.45

Leucine 2.16 Valine 1.43

Inorganics (%)

Calcium 0.020 Phosphate 3.848

Chloride 9.326 Potassium 4.183

Cobalt <0.001 Sodium 9.351

Copper 0.001 Sulfate 1.004

Iron 0.003 Sulfur 1.629

Lead <0.001 Tin <0.001

Magnesium 0.017 Zinc 0.002

Manganese <0.001

Vitamins (µg/g)

Biotin 0.3 PABA 95.0

Choline

(as Choline Chloride)

4170.7 Pantothenic Acid 63.2

Cyanocobalamin 0.3 Pyridoxine 7.2

Folic Acid 1.5 Riboflavin 26.8

Inositol 2820.0 Thiamine NA

Nicotinic Acid 603.0 Thymidine 55.0

Biological Testing (CFU/g)

Coliform negative Standard Plate Count 18

Salmonella negative Thermophile Count 379

Precautions

1. For Laboratory Use.

2. Follow proper established laboratory procedures in handling and

disposing of infectious materials.

Storage

Store the dehydrated product below 30°C. The dehydrated product is

very hygroscopic. Keep container tightly closed.

Expiration Date

The expiration date applies to the product in its intact container when

stored as directed. Do not use a product if it fails to meet specifications

for identity and performance.

Procedure

Materials Provided

Fish Peptone No. 1

Materials Required But Not Provided

Materials vary depending on the medium being prepared.

Method of Preparation

Refer to the final concentration of peptone in the formula of the

medium being prepared. Add Fish Peptone No. 1 as required.

Specimen Collection and Preparation

Obtain and process specimens according to the techniques and

procedures established by laboratory policy.

Test Procedure

See appropriate references for specific procedures on the medium

being prepared or the sample being analyzed.

Results

Refer to appropriate references and procedures for results.

Limitations of the Procedure

1. Since the nutritional requirements of organisms vary, some strains may

be encountered that fail to grow or grow poorly on prepared medium.

Packaging

Fish Peptone No. 1 500 g 0551-17

10 kg 0551-08

The Difco Manual 197

Section II Fletcher Medium Base

Bacto

Fletcher Medium Base

Intended Use

Bacto Fletcher Medium Base is used with sterile normal rabbit serum

for isolating and cultivating Leptospira.

Summary and Explanation

In 1816, Adolf Weil described the first recognized infections of

Leptospirosis in humans.

These cases were caused by Leptospira

interogans serovar icterohaemorrhagiae and the disease was subse-

quently named Weil’s Disease.

Leptospirosis is a zoonotic disease,

having its reservoir in wild, domestic, and peridomestic animals.

Infection usually results from direct or indirect exposure to the urine

of leptospiruric animals.

Indirect exposure through contaminated

water and soil accounts for most sporadic cases.

Direct exposure occurs

in pet owners, veterinarians and persons working with livestock.

Leptospirosis is typically a biphasic illness.

3,8

The infection is acute in

onset, with a flu- like syndrome persisting for 4 to 7 days.

Onset of a

second “immune” phase, in which meningitis, skin rash, and hepatic

and renal involvement may be present, occurs within a few days.

Fletcher Medium Base is prepared according to the formulation of

Fletcher.

Myers et al.

reported Fletcher Medium, with a basal agar

layer containing charcoal, to be superior to the standard medium for

the maintenance of leptospiral cultures. Fletcher Medium Base

prepared with sterile normal rabbit serum is specified for the isolation

of Leptospira.

3,4,7

Principles of the Procedure

Bacto Peptone and Beef Extract provide the nitrogen, vitamins, carbon

and amino acids in Fletcher Medium Base. Sodium chloride maintains

the osmotic balance of the medium. Bacto Agar is the solidifying agent.

Sterile normal rabbit serum is added to the formula to stimulate growth

of Leptospira.

Formula

Fletcher Medium Base

Formula Per Liter

Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3 g

Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g

Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g

Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g

Final pH 7.9 ± 0.1 at 25°C

Precautions

1. For Laboratory Use.

2. Follow proper established laboratory procedures in handling and

disposing of infectious materials.

3. Leptospira species are BioSafety Level 2 pathogens. Handling clini-

cal specimen material potentially infected with Leptospira species

should be performed in a Class II biological safety cabinet (BSC).

Storage

Store the dehydrated medium below 30°C. The dehydrated medium is

very hygroscopic. Keep container tightly closed.

Expiration Date

The expiration date applies to the product in its intact container when

stored as directed. Do not use a product if it fails to meet specifications

for identity and performance.

Procedure

Materials Provided

Fletcher Medium Base

Materials Required But Not Provided

Glassware

Autoclave

Incubator (35°C)

Waterbath (56°C)

Sterile normal rabbit serum

Method of Preparation

1. Suspend 2.5 grams in 920 ml distilled or deionized water.

2. Heat to boiling to dissolve completely.

3. Autoclave at 121°C for 15 minutes. Cool to 56°C.

4. Aseptically add 80 ml sterile normal rabbit serum at 56°C. Mix well.

5. Determine pH. If necessary, aseptically adjust to pH 7.9 ± 0.1 with

1 N HCl or 1 N NaOH.

Specimen Collection and Preparation

Obtain and process specimens according to the techniques and procedures

established by laboratory policy. Blood, cerebrospinal fluid (CSF) and

User Quality Control

Identity Specifications

Dehydrated Appearance: Beige, free-flowing, homogeneous.

Solution: 2.5 g in 920 ml distilled or deionized

water, soluble upon boiling. Solution

is very light amber, clear to very

slightly opalescent without

significant precipitate.

Prepared Medium: Very light amber, very slightly to

slightly opalescent without

significant precipitate.

Reaction of 2.5 g in

920 ml distilled water: pH 7.9 ± 0.1 at 25°C

Cultural Response

Prepare Fletcher Medium Base per label directions. Enrich

with sterile normal rabbit serum. Inoculate and incubate at

30 ± 2°C for up to 5 days.

ORGANISM ATCC

INOCULUM GROWTH

Leptospira interrogans 23605 2-3 drops good

serovar australis

Leptospira interrogans 23470 2-3 drops good

serovar canicola

Leptospira kirschneri 23604 2-3 drops good

serovar grippotyphosa

The cultures listed are the minimum that should be used for

performance testing.

198 The Difco Manual

2. Myers, D. M., V. M. Varela-Diaz, and A. A. Siniuk. 1973.

Long-term survival of Leptospira in a biphasic culture medium

containing charcoal. Am. Soc. Microbiol. 25:514-516.

3. Kaufmann, A. F., and R. S. Weyant. 1995. Leptospiraceae,

p. 621-625. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover

and R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed.

American Society for Microbiology, Washington, D.C.

4. Koneman, E. W., S. D. Allen, V. R. Dowell, Jr., W. M. Janda,

H. M. Sommers, and W. C. Winn, Jr. 1988. Color atlas and

textbook of diagnostic microbiology, 3rd ed. J. B. Lippincott

Company, Philadelphia, PA.

5. Elliott, S. H. 1980. Discussion and clinical diagnosis of

Leptospirosis. J. Am. Med. Tech. 42:37-44.

6. Faine, S. (ed.). 1982. Guidelines for the control of Leptospirosis. W.

H. O. Offset publication no. 67. World Health Organization, Geneva.

7. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures

handbook. American Society for Microbiology, Washington, D.C.

8. Kelley, P. W. 1992. Leptospirosis, p. 1295-1301. In S. L. Gorbach,

J. G. Bartlett, and N. R. Blacklow (ed.), Infectious diseases. The

W. B. Saunders Co., Philadelphia, PA.

9. Weinman, D. 1981. Bartonellosis and anemias associated with

bartonella-like structures, p. 235-248. In A. Balows, and W. J.

Hausler, Jr. (ed.), Diagnostic procedures for bacterial, mycotic and

parasitic infections, 6th ed. American Public Health Association,

Washington, D.C.

Packaging

Fletcher Medium Base 500 g 0987-17

urine are the specimens of choice for the recovery of leptospires from

patients with leptospirosis.

Test Procedure

1. Aseptically dispense into sterile screw-cap tubes in 5-7 ml amounts.

Store at room temperature overnight.

2. Inactivate the whole medium the day following its preparation by

placing the tubes in a water bath at 56°C for 1 hour.

3. Allow the medium to cool before inoculation.

4. Growth is first seen in approximately 10 days at 35°C (2 to 4 weeks

at 25°C) as a cloud of minute granules that develop into

microcolonies just below the surface.

5. Gram stain is not satisfactory. The microcolonies can be fixed

with methanol and stained with Giemsa stain to show rod forms

at the edges.

Results

Refer to appropriate references and procedures for results.

Limitations of the Procedure

1. Since the nutritional requirements of organisms vary, some

strains may be encountered that fail to grow or grow poorly on

this medium.

References

1. Fletcher, W. 1928. Recent work on leptospirosis, tsutsugamushi

disease and tropical typhus in the federated Malay States. Trans.

Roy. Soc. Trop. Med. Hyg. 21: 265-287.

Folic AOAC Medium Section II

Bacto

Folic AOAC Medium

User Quality Control

Identity Specifications

Dehydrated Appearance: Off-white, free-flowing,

homogeneous.

Solution: 5.50% solution (single strength) and

11.0% solution (double strength),

soluble in distilled or deionized

water on boiling.

Solution is light amber, clear, may

have a slight precipitate.

Prepared Medium: Very light amber, clear.

Reaction of 5.50%

Solution at 25°C: pH 6.7 ± 0.1

Cultural Response

Prepare Folic AOAC Medium per label directions. This

medium should support the growth of E. hirae ATCC

8043

when prepared in single strength and supplemented with

folic acid.

Intended Use

Bacto Folic AOAC Medium is used for determining folic acid concen-

tration by the microbiological assay technique.

Also Known As

AOAC is an abbreviation for Association of Official Analytical Chemists.

Summary and Explanation

Vitamin Assay Media are prepared for use in the microbiological assay

of vitamins. Three types of media are used for this purpose:

1. Maintenance Media: For carrying the stock culture to preserve

the viability and sensitivity of the test organism for its intended

purpose.

2. Inoculum Media: To condition the test culture for immediate use.

3. Assay Media: to permit quantitation of the vitamin under test.

Folic AOAC Medium is prepared for use in the microbiological assay

of folic acid according to the procedures of the Folic Acid Assay in

AOAC.

Enterococcus hirae ATCC

8043 (Streptococcus faecium) is

the test organism in this assay.

Principles of the Procedure

Folic Acid AOAC Medium is a folic acid-free dehydrated medium

containing all other nutrients and vitamins essential for the cultivation

of E. hirae ATCC

8043. The addition of folic acid in specified

The Difco Manual 199

Section II Folic AOAC Medium

increasing concentrations gives a growth response that can be

measured turbidimetrically or titrimetrically.

Formula

Folic AOAC Medium

Formula Per Liter

Bacto Vitamin Assay Casamino Acids. . . . . . . . . . . . . . . . 10 g

L-Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 g

L-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g

L-Cysteine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . 0.76 g

Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g

Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg

Guanine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg

Uracil. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg

Xanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg

p-Aminobenzoic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg

Pyridoxine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mg

Thiamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . 400 µg

Calcium Pantothenate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800 µg

Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800 µg

Biotin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 µg

Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg

Glutathione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 mg

Sorbitan Monooleate Complex. . . . . . . . . . . . . . . . . . . . . . 0.1 g

Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 g

Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 g

Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 g

Manganese Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg

Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg

Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg

Final pH 6.7 ± 0.1 at 25°C

Precautions

1. For Laboratory Use.

2. Follow proper established laboratory procedures in handling and

disposing of infectious materials.

3. Great care must be taken to avoid contamination of media or

glassware in microbiological assay procedures. Extremely small

amounts of foreign material may be sufficient to give erroneous

results. Scrupulously clean glassware free from detergents and

other chemicals must be used. Glassware is heated to 250°C for at

least 1 hour to burn off any organic residues that might be present.

4. Take precautions to keep sterilizing and cooling conditions uniform

throughout the assay.

Storage

Store the dehydrated medium at 2-8°C. The dehydrated medium is very

hygroscopic. Keep container tightly closed.

Expiration Date

The expiration date applies to the product in its intact container when

stored as directed. Do not use a product if it fails to meet specifications

for identity and performance.

Procedure

Materials Provided

Folic AOAC Medium

Materials Required But Not Provided

Glassware

Autoclave

Stock culture of Enterococcus hirae ATCC

8043

Sterile tubes

Distilled or deionized water

Folic Acid

0.01 N NaOH

Dilute HCl

Spectrophotometer or Nephelometer

Method of Preparation

1. Suspend 11 grams in 100 ml distilled or deionized water.

2. Heat to boiling for 2-3 minutes.

3. Distribute 5 ml amounts into tubes, evenly dispersing the precipitate.

4. Add standard or test samples.

5. Adjust tube volume to 10 ml with distilled or deionized water.

6. Autoclave at 121°C for 5 minutes.

Specimen Collection and Preparation

Assay samples are prepared according to references given in the

specific assay procedures. For assays, the samples should be diluted to

approximately the same concentration as the standard solution.

Test Procedure

Follow assay procedures as outlined in AOAC.

It is essential that a

standard curve be set up for each separate assay. Autoclaving and

incubation conditions that can influence the standard curve readings

cannot always be duplicated. The standard curve is obtained by using

folic acid at levels of 0.0, 1, 2, 4, 6, 8 and 10 ng per assay tube (10 ml).

Folic AOAC Medium may be used for both turbidimetric and titrimetric

analysis. Turbidimetric readings should be taken after incubation at

35-37°C for 16-18 hours. Titrimetric determinations are best made

following incubation at 35-37°C for 72 hours.

The folic acid required for the preparation of the standard curve may

be prepared as follows:

A. Dissolve 50 mg dried folic acid in about 30 ml 0.01N NaOH

and 300 ml distilled water.

B. Adjust the pH reaction to 7.5 ± 0.5 with diluted HCl solution.

Dilute to 500 ml with distilled water.

C. Add 2 ml of the solution to 50 ml distilled water. Adjust the

pH reaction to 7.5 ± 0.5. Dilute to 100 ml with distilled water.

This yields a stock solution containing 2 mcg folic acid per ml.

D. Prepare the stock solution fresh daily.

The standard solution for the assay is made by diluting 1 ml of this

stock solution to 1 liter with distilled water. This solution contains 2 ng

folic acid per ml. Use 0.0, 0.5, 1, 2, 3, 4, and 5 ml per assay tube.

Some laboratories may wish to alter the concentration of folic acid

recommended above for the standard curve. This is permissible if the

concentration used is within the limits specified by AOAC.

Results

1. Prepare a standard concentration response curve by plotting the

response readings against the amount of standard in each tube,

disk or cup.

200 The Difco Manual

2. Determine the amount of vitamin at each level of assay solution by

interpolation from the standard curve.

3. Calculate the concentration of vitamin in the sample from the

average of these volumes. Use only those values that do not vary

more than ±10% from the average. Use the results only if two thirds

of the values do not vary more than ±10%.

Limitations of the Procedure

1. The test organism used for inoculating an assay medium must

be cultured and maintained on media recommended for this

purpose.

2. Aseptic technique should be used throughout the assay procedure.

Bacto

Folic Acid Assay Medium

User Quality Control

Identity Specifications

Dehydrated Appearance: Off white to very light beige,

free-flowing, homogeneous.

Solution: 3.75% (single strength) or 7.5%

(double strength) solution, soluble

in distilled or deionized water upon

boiling for 2-3 minutes. Solution is

light amber, clear, may have a slight

precipitate.

Prepared Medium: Very light amber, clear, may have a

very slight precipitate.

Reaction of 3.75%

Solution at 25°C: pH 6.8 ± 0.2

Cultural Response

Prepare single-strength Folic Acid Assay Medium per label

directions. The medium should support the growth of E. hirae

ATCC

8043. The most effective range is 2-10 ng folic acid

per 10 ml tube.

Intended Use

Bacto Folic Acid Assay Medium is used for determining folic acid

concentration by the microbiological assay technique.

Summary and Explanation

Vitamin Assay Media are prepared for use in the microbiological assay

of vitamins. Three types of medium are used for this purpose:

1. Maintenance Medium: For carrying the stock culture to preserve the

viability and sensitivity of the test organism for its intended purpose.

2. Inoculum Medium: To condition the test culture for immediate use.

3. Assay Medium: To permit quantitation of the vitamin under test.

Folic Acid Assay Medium is used in the microbiological assay of folic

acid with Enterococcus hirae ATCC

8043 as the test organism. Folic

Acid Assay Medium is prepared according to the formula described by

Capps, Hobbs and Fox,

modified with sodium citrate instead of

sodium acetate.

Principles of the Procedure

Folic Acid Assay Medium is a folic acid-free dehydrated medium

containing all other nutrients and vitamins essential for the cultivation

of E. hirae ATCC

8043. The addition of folic acid in specified

increasing concentrations gives a growth response that can be measured

turbidimetrically.

Formula

Folic Acid Assay Medium

Formula Per Liter

Bacto Vitamin Assay Casamino Acids. . . . . . . . . . . . . . . . 12 g

Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g

Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g

L-Cystine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g

DL-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g

Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg

Guanine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg

Uracil. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg

Thiamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mg

Pyridoxine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mg

Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mg

Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mg

p-Aminobenzoic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 µg

Biotin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8 µg

Calcium Pantothenate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400 µg

Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g

Monopotassium Phosphate. . . . . . . . . . . . . . . . . . . . . . . . . . 1 g

Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 g

Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg

Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg

Manganese Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg

Final pH 6.8 ± 0.2 at 25°C

Precautions

1. For Laboratory Use.

2. Follow proper established laboratory procedures in handling and

disposing of infectious materials.

3. Great care must be taken to avoid contamination of media or glass-

ware in microbiological assay procedures. Extremely small

Folic Acid Assay Medium Section II

3. The use of altered or deficient media may cause mutants

having different nutritional requirements that will not give a

satisfactory response.

4. For successful results of these procedures, all conditions of the

assay must be followed precisely.

References

1. Association of Official Analytical Chemists. 1995. Official

methods of analysis of AOAC international, 16th ed. AOAC

International, Arlington, VA.

Packaging

Folic AOAC Medium 100 g 0967-15*

*Store at 2-8°C

The Difco Manual 201

amounts of foreign material may be sufficient to give erroneous

results. Scrupulously clean glassware free from detergents and

other chemicals must be used. Glassware must be heated to 250°C

for at least 1 hour to burn off any organic residues that might be

present.

4. Take precautions to keep sterilization and cooling conditions

uniform throughout the assay.

Storage

Store the dehydrated medium at 2-8°C. The dehydrated medium is very

hygroscopic. Keep container tightly closed.

Expiration Date

The expiration date applies to the product in its intact container when

stored as directed. Do not use a product if it fails to meet specifications

for identity and performance.

Procedure

Materials Provided

Folic Acid Assay Medium

Materials Required But Not Provided

Glassware

Autoclave

Stock culture of Enterococcus hirae ATCC

8043

Sterile tubes

Sterile 0.85% saline

Distilled or deionized water

0.01 N NaOH

Dilute HCl

Folic Acid USP

Lactobacilli Agar AOAC

Lactobacilli Broth AOAC

Centrifuge

Spectrophotometer

Method of Preparation

1. Suspend 7.5 grams in 100 ml distilled or deionized water.

2. Boil 2-3 minutes to dissolve completely.

3. Dispense 5 ml amounts into tubes, evenly dispersing the precipitate.

4. Add standard or test samples.

5. Adjust tube volume to 10 ml with distilled or deionized water.

6. Autoclave at 121°C for 10 minutes.

Specimen Collection and Preparation

Prepare assay samples according to references given in the specific

assay procedures. Dilute the samples to approximately the same

concentration as the standard solution.

Test Procedure

Prepare stock cultures of E. hirae ATCC

8043 by stab inoculation of

Lactobacilli Agar AOAC. Incubate at 35-37°C for 24-48 hours. Store

tubes in the refrigerator. Make transfers at monthly intervals. Prepare

the inoculum for assay by subculturing a stock culture of E. hirae

ATCC

8043 into a tube containing 10 ml of Lactobacilli Broth AOAC.

After incubation at 35-37°C for 18-24 hours, centrifuge the cells under

aseptic conditions and decant the supernatant. Wash the cells three

times with 10 ml of sterile 0.85% saline. After the third wash, dilute

the cell suspension 1:100 with sterile 0.85% saline. Use one drop of

this latter suspension to inoculate each of the assay tubes.

It is essential that a standard curve be set up for each separate assay.

Autoclaving and incubation conditions that influence the standard

curve readings cannot always be duplicated. The standard curve is

obtained by using folic acid at levels of 0.0, 2, 4, 6, 8 and 10 ng per

10 ml assay tube. Turbidimetric readings should be made after

incubation at 35-37°C for 18-24 hours. Refrigerate tubes for 15-30

minutes to stop growth before reading.

Prepare the folic acid stock solution required for the standard curve as

follows:

1. Dissolve 50 mg dried Folic Acid USP Reference Standard or

equivalent in about 30 ml of 0.01 N NaOH and 300 ml distilled

water.

2. Adjust to pH 7.5 ± 0.5 with diluted HCl solution. Add distilled

water to give a volume of 500 ml.

3. Add 2 ml of the solution from step 2 to 50 ml distilled water.

Adjust the pH to 7.5 ± 0.5 with HCl solution. Dilute to 100 ml with

distilled water to give a stock solution containing 2 mcg folic acid

per ml. Prepare the stock solution fresh daily.

Prepare the standard solution for the assay by diluting 1 ml of this

stock solution in 1 liter with distilled water. This solution contains 2 ng

folic acid per ml. Use 0.0, 0.5, 1, 2, 3, 4 and 5 ml per assay tube.

Following incubation, place the tubes in the refrigerator for 15-30

minutes to stop growth. The growth can be measured by a turbidimetric

method and the curve constructed from the values obtained. The most

effective assay range is between the levels of 2 and 10 ng folic acid per

10 ml tube.

Results

1. Prepare a standard concentration response curve by plotting the

response readings against the amount of standard in each tube, disk

or cup.

2. Determine the amount of vitamin at each level of assay solution by

interpolation from the standard curve.

3. Calculate the concentration of vitamin in the sample from the

average of these volumes. Use only those values that do not vary

more than ±10% from the average. Use the results only if two thirds

of the values do not vary more than ±10%.

Limitations of the Procedure

1. The test organism used for inoculating an assay medium must be

cultured and maintained on media recommended for this purpose.

2. Aseptic technique should be used throughout the assay procedure.

3. The use of altered or deficient media may cause mutants having

different nutritional requirements that will not give a satisfactory

response.

4. For successful results of these procedures, all conditions of the

assay must be followed precisely.

References

1. Capps, Hobbs, and Fox. 1948. J. Bacteriol. 55:869.

Packaging

Folic Acid Assay Medium 100 g 0318-15

Section II Folic Acid Assay Medium

202 The Difco Manual

Folic Acid Casei Medium & Folic Buffer A, Dried Section II

Bacto

Folic Acid Casei Medium

Bacto Folic Buffer A, Dried

User Quality Control

Identity Specifications

Folic Acid Casei Medium

Dehydrated Appearance: Off-white, homogeneous, with a

tendency to clump.

Solution: 4.7% (single strength) and 9.4%

(double strength) solution, soluble

in distilled or deionized water upon

boiling 1-2 minutes. Single-strength

solution is light amber, clear, may

have a slight precipitate.

Prepared Medium: Single-strength solution is very light

amber, clear, may have a very slight

precipitate.

Reaction of 4.7%

Solution at 25°C: 6.7 ± 0.1

Folic Buffer A, Dried

Dehydrated Appearance: White to off-white, free-flowing,

homogeneous.

Solution: 1.54% solution, soluble in distilled

or deionized water.

Solution Appearance: Colorless to very light amber, clear.

Reaction of 1.54%

Solution at 25°C: pH 6.1 ± 0.05

Cultural Response

Prepare Folic Acid Casei Medium per label directions. The

medium is tested by creating a standard curve using Folic Acid

at concentrations of 0 to 1.0 ng per 10 ml. This medium should

support the growth of L. casei subsp. rhamnosus ATCC

7469

when prepared in single strength and supplemented with

ascorbic acid and Folic Acid.

Intended Use

Bacto Folic Acid Casei Medium is used for determining folic acid

concentration by the microbiological assay technique.

Bacto Folic Buffer A, Dried is prepared for use with Folic Acid Casei

Medium in the microbiological assay of serum folic acid.

Summary and Explanation

Vitamin Assay Media are prepared for use in the microbiological assay

of vitamins. Three types of media are used for this purpose:

1. Maintenance Media: For carrying the stock culture to preserve the

viability and sensitivity of the test organism for its intended purpose;

2. Inoculum Media: To condition the test culture for immediate use;

3. Assay Media: To permit quantitation of the vitamin under test.

Folic Acid Casei Medium is prepared for the microbiological assay of

folic acid, particularly folic acid in serum. Lactobacillus casei subsp.

rhamnosus ATCC

7469 is used as the test organism in this assay.

Folic Acid Casei Medium is prepared according to the formulation

described by Flynn, Williams, O’Dell and Hogan

and modified by

Baker et al.

and Waters and Mollin.

Total serum folic acid activity can vary depending on the disease state.

It has been reported that normal subjects have a mean serum folic acid

level of 9.9 ng per ml. Patients with uncomplicated pernicious anemia

have a mean serum folic acid level of 16.6 ng per ml while patients

with megaloblastic anemia have levels less than 4.0 ng per ml.

Folic Buffer A, Dried is used for preparing both the standard and the

serum specimen in the microbiological assay of folic acid.

Principles of the Procedure

Folic Acid Casei Medium is a folic acid-free dehydrated medium

containing all other nutrients and vitamins essential for the cultivation

of L. casei subsp. rhamnosus ATCC

7469. The addition of folic acid

in specified increasing concentrations gives a growth response that can

be measured turbidimetrically.

Formula

Folic Acid Casei Medium

Formula Per Liter

Charcoal Treated Casitone . . . . . . . . . . . . . . . . . . . . . . . . . 10 g

Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g

Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g

Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g

Monopotassium Phosphate. . . . . . . . . . . . . . . . . . . . . . . . . . 1 g

DL-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 g

L-Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6 g

L-Cysteine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g

Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg

Guanine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg

Uracil. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg

Xanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg

Sorbitan Monooleate Complex. . . . . . . . . . . . . . . . . . . . . . 0.1 g

Glutathione (reduced) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mg

Magnesium Sulfate, Anhydrous . . . . . . . . . . . . . . . . . . . . . 0.2 g

Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg

Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg

Manganese Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 mg

Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mg

p-Aminobenzoic Acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 mg

Pyridoxine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mg

Thiamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . 400 µg

Calcium Pantothenate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800 µg

Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800 µg

Biotin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 µg

Final pH 6.7 ± 0.1

Folic Buffer A, Dried

Formula Per Liter

Monopotassium Phosphate. . . . . . . . . . . . . . . . . . . . . . 10.656 g

Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . 3.744 g

Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g

Final pH 6.1 ± 0.05

The Difco Manual 203

Section II Folic Acid Casei Medium & Folic Buffer A, Dried

Precautions

1. For Laboratory Use.

2. Great care must be taken to avoid contamination of media or glass-

ware in microbiological assay procedures. Extremely small

amounts of foreign material may be sufficient to give erroneous

results. Scrupulously clean glassware free from detergents and

other chemicals must be used. Glassware must be heated to 250°C

for at least 1 hour to burn off any organic residues that might be present.

3. Take precautions to keep sterilization and cooling conditions

uniform throughout the assay.

4. Follow proper established laboratory procedures in handling and

disposing of infectious materials.

Storage

Store Folic Acid Casei Medium and Folic Buffer A, Dried at 2-8°C.

The dehydrated medium is very hygroscopic. Keep container tightly

closed.

Expiration Date

The expiration date applies to the product in its intact container when

stored as directed. Do not use a product if it fails to meet specifications

for identity and performance.

Procedure

Materials Provided

Folic Acid Casei Medium

Folic Buffer A, Dried

Materials Required But Not Provided

Glassware

Autoclave

Stock culture of Lactobacillus casei subsp. rhamnosus ATCC

7469

Ascorbic acid (25 mg)

Sterile 0.85% saline

Distilled and deionized water

0.01 N NaOH

0.05 N HCl

Lactobacilli Agar AOAC

Folic Acid

Incubator (35-37°C)

Micro Inoculum Broth

Centrifuge

Spectrophotometer

Method of Preparation

Folic Acid Casei Medium

1. Suspend 9.4 grams in 100 ml distilled or deionized water.

2. Add 50 mg ascorbic acid if standard and test samples are not prepared

in Folic Buffer A.

3. Boil for 1-2 minutes.

4. Dispense 5 ml amounts into tubes, evenly dispersing any precipitate.

5. Add standard or test samples.

6. Adjust tube volume to 10 ml with distilled or deionized water.

7. Autoclave at 121°C for 5 minutes.

Folic Buffer A Dried

1. Dissolve the contents of one vial (15.4 grams) in 1 liter distilled or

deionized water.

Specimen Collection and Preparation

Assay samples are prepared according to references given in the

specific assay procedures. For assays, the samples should be diluted to

approximately the same concentration as the standard solution.

Test Procedure

Preparation of Stock Cultures and Inoculum

Prepare stock cultures of the test organism, L. casei subsp. rhamnosus

ATCC

7469, by stab inoculation into prepared tubes of Lactobacilli Agar

AOAC. Incubate the cultures at 35-37°C for 18-24 hours. Store cultures

in the refrigerator at 2-8°C. Stock transfers are made at monthly intervals.

Prepare the inoculum for assay by subculturing from a stock culture of

L. casei subsp. rhamnosus into a tube containing 10 ml prepared

Micro Inoculum Broth. Incubate at 35-37°C for 16-18 hours. Under

aseptic conditions, centrifuge the tubes to sediment the cells and decant

the supernatant. Wash the cells in 10 ml sterile single-strength Folic

Acid Casei Medium. Resediment the cells by centrifuging aseptically

and decant the supernatant. Repeat washing two more times. After the

third washing, resuspend the cells in 10 ml sterile single-strength

medium and dilute 1 ml with 99 ml of the same medium. One drop of

this suspension is used to inoculate each of the assay tubes. Read the

growth response of the assay tubes turbidimetrically after 18-24 hours

incubation at 35-37°C. (Some laboratories use 0.85% saline instead of

the single-strength basal medium to wash and dilute the inoculum.)

Preparation of the Standard

It is essential that a standard curve be constructed for each separate

assay. Autoclave and incubation conditions can influence the standard

curve readings and cannot always be duplicated. The standard curve

may be obtained by using folic acid at levels of 0.0, 0.1, 0.2, 0.4, 0.6,

0.8 and 1 ng per assay tube (10 ml).

The folic acid required for preparation of the standard curve may be

prepared as follows:

Dissolve 50 mg dried folic acid in about 30 ml 0.01 N NaOH and 300

ml distilled water. Adjust to pH 7-8 with 0.05 N HCl and dilute to 500

ml with distilled water. Dilute 10 ml of this solution with 500 ml

distilled water. Further dilute 1 ml in 1 liter distilled water to make a

stock solution containing 2 ng per ml folic acid. Prepare the standard

solution containing 0.2 ng per ml folic acid by diluting 10 ml of stock

solution with 90 ml of Folic Buffer A, Dried solution. Use 0.0, 0.5, 1,

2, 3, 4 and 5 ml per assay tube.

Prepare the stock solution fresh daily.

Preservation of Serum Specimens

1. Allow the blood specimen to clot and the serum to separate from

the clot.

2. Aspirate the serum into a clean dry tube and centrifuge to remove

any cells that may be present. Avoid hemolysis. Dispense 5 ml

of each serum sample into clean dry test tubes and add 25 mg

ascorbic acid to each tube.

3. If the test is not begun immediately, place tubes in a freezer and

hold below -20°C.

204 The Difco Manual

Preparation of Serum Specimen

1. Thaw the serum containing ascorbic acid.

2. Add 5 ml of the uniform sample to 45 ml rehydrated Folic Buffer

A, Dried.

3. Incubate the serum-buffer solution at 37°C for 90 minutes. Autoclave

the incubated mixture at 121°C for 2.5 minutes.

4. Remove the coagulated protein by centrifuging and transfer the

clear supernatant to a clean dry tube. The clear solution is the

sample to use in the folic acid assay.

Procedure for Total Folic Acid

1. Use 0.5, 1.0, 1.5 ml or other volumes of the prepared serum

extracts as described above.

2. Fill each assay tube with 5 ml of rehydrated Folic Acid Casei

Medium and sufficient distilled or deionized water to give a total

volume of 10 ml per tube.

3. Autoclave tubes at 121°C for 5 minutes.

4. Add 1 drop of inoculum described under Preparation of Stock

Culture and Inoculum to each assay.

5. Incubate at 35-37°C for 18-24 hours. Tubes are refrigerated for

15-30 minutes to stop growth before reading turbidimetrically.

Results

The amount of folic acid in the test samples can be determined by

Fraser Broth Section II

Fraser Broth

Bacto

Fraser Broth Base

Fraser Broth Supplement

Intended Use

Bacto Fraser Broth Base is used with Bacto Fraser Broth Supplement

in selectively enriching and detecting Listeria.

Summary and Explanation

First described in 1926 by Murray, Webb and Swann,

Listeria

monocytogenes is a widespread problem in public health and the food

industries. This organism has the ability to cause human illness

and death, particularly in immunocompromised individuals and

pregnant women.

The first reported food-borne outbreak of listeriosis

was in 1985,

and since then, microbiological and epidemiological

evidence from both sporadic and epidemic cases of listeriosis has

indicated that the principle route of transmission is via the consumption

of foodstuffs contaminated with Listeria monocytogenes.

Implicated vehicles of transmission include turkey frankfurters,

coleslaw, pasteurized milk, Mexican-style cheese, paté, and pickled pork

tongue. The organism has been isolated from commercial dairy and other

food processing plants, and is ubiquitous in nature, being present in a

wide range of unprocessed foods as well as in soil, sewage, silage

and river water.

Bacto Fraser Broth Base and Bacto Fraser Broth Supplement are based

on the formulation of Fraser and Sperber.

The medium is use in

the rapid detection of Listeria from food

and environmental samples.

Many common food contaminants such as streptococci, enterococci,

Bacillus species, Escherichia coli, Pseudomonas aeruginosa and Proteus

vulgaris interfere with the isolation of Listeria monocytogenes.

Listeria species grow over a pH range of 5.0-9.6, and survive in food

products with pH levels outside these parameters.

Listeria spp. are

microaerophilic, gram-positive, asporogenous, non-encapsulated, non-

branching, regular, short, motile rods. Motility is most pronounced at 20°C.

Identification of Listeria is based on successful isolation of the

organism, biochemical characterization and serological confirmation.

Principles of the Procedure

Bacto Tryptose, Bacto Beef Extract and Bacto Yeast Extract provide

nitrogen, vitamins and minerals. Sodium phosphate and potassium

phosphate are buffering agents. Differentiation is aided by including

ferric ammonium citrate in the final medium. Since all Listeria species

hydrolyze esculin, the addition of ferric ions to the medium will detect

the reaction. A blackening of the medium by cultures containing

esculin-hydrolyzing bacteria is the result of the formation of

6,7-dihydroxycoumarin that reacts with the ferric ions.

Selectivity is provided by the presence of lithium chloride, nalidixic

acid and acriflavine in the formula. The high salt tolerance of Listeria

is used as a means to inhibit growth of enterococci.

interpolating the results with the values obtained on the standard curve,

taking into consideration the dilutions of the samples.

Limitations of the Procedure

1. The test organism used for inoculating an assay medium must

be cultured and maintained on media recommended for this

purpose.

2. Aseptic technique should be used throughout the assay procedure.

3. The use of altered or deficient media may cause mutants having

different nutritional requirements that will not give a satisfactory

response.

4. For successful results of these procedures, all conditions of the

assay must be followed precisely.

References

1. Flynn, Williams, O’Dell, and Hogan. 1951. Anal. Chem. 23:180.

2. Baker, Herbert, Frank, Pasher, Hunter, Wasserman, and

Sobotka. 1959. Clin. Chem. 5:275.

3. Waters and Molin. 1961. J. Clin. Pathol. 14:335.

Packaging

Folic Acid Casei Medium 100 g 0822-15

Folic Buffer A, Dried 6 x 15.4 g 3246-33

The Difco Manual 205

Section II Fraser Broth

Listeria monocytogenes

ATCC

19114

Uninoculated

tube

User Quality Control

Identity Specifications

Fraser Broth Base

Dehydrated Appearance: Tan, free-flowing, homogeneous.

Solution: 5.5% solution, soluble in distilled or deionized water on

boiling. Solution is medium amber, clear to slightly

opalescent with a fine precipitate.

Prepared Tubes: Medium amber, clear to slightly opalescent with

a fine precipitate.

Reaction of 5.5%

Solution at 25°C: pH 7.2 ± 0.2

Bacto Fraser Broth Supplement

Solution Appearance: Dark brown solution.

Cultural Response

Prepare Fraser Broth Base per label directions. Add Fraser Broth Supplement.

Inoculate and incubate at 35 ± 2°C for 24-48 hours.

INOCULUM ESCULINE

ORGANISM ATCC

CFU GROWTH REACTION

Escherichia faecalis 29212* 1,000-2,000 marked to complete inhibition –

Escherichia coli 25922* 1,000-2,000 marked to complete inhibition –

Listeria monocytogenes 19114 100-1,000 good +

The cultures listed are the minimum that should be used for performance testing.

*These cultures are available as Bactrol™ Disks and should be used as directed in Bactrol Disks Technical Information.

Formula

Fraser Broth Base

Formula Per Liter

Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g

Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g

Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g

Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g

Sodium Phosphate, Dibasic . . . . . . . . . . . . . . . . . . . . . . . . 9.6 g

Potassium Phosphate, Monobasic . . . . . . . . . . . . . . . . . . 1.35 g

Esculin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g

Nalidixic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02 g

Acriflavine HCl. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.024 g

Lithium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g

Final pH 7.2 ± 0.2 at 25°C

Fraser Broth Supplement

Ingredients per 10 ml vial

Ferric Ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g

Precautions

1. For Laboratory Use.

2. Fraser Broth Base:

HARMFUL. IRRITATING TO EYES, RESPIRATORY SYSTEM

AND SKIN. MAY CAUSE HARM TO THE UNBORN CHILD.

Avoid contact with skin and eyes. Do not breathe dust. Wear

suitable protective clothing. Keep container tightly closed.

TARGET ORGAN(S): Blood, Kidneys, Nerves

FIRST AID: In case of contact with eyes, rinse immediately with

plenty of water and seek medical advice. After contact with skin,

wash immediately with plenty of water. If inhaled, remove to fresh

air. If not breathing, give artificial respiration. If breathing is

difficult, give oxygen. Seek medical advice. If swallowed seek

medical advice immediately and show this container or label.

Fraser Broth Supplement:

IRRITANT. IRRITATING TO EYES, RESPIRATORY SYSTEM

AND SKIN. Avoid contact with skin and eyes. Do not breathe mist.

Wear suitable protective clothing. Keep container tightly closed.

FIRST AID: In case of contact with eyes, rinse immediately with

plenty of water and seek medical advice. After contact with skin,

wash immediately with plenty of water. If inhaled, remove to fresh

air. If not breathing, give artificial respiration. If breathing is

difficult, give oxygen. Seek medical advice. If swallowed seek

medical advice immediately and show this container or label.

3. Follow proper, established laboratory procedures in handling and

disposing of infectious materials.

Storage

Store the dehydrated medium below 30°C. The dehydrated medium is

very hygroscopic. Keep container tightly closed.

Store Bacto Fraser Broth Supplement at 2-8°C.

Store the prepared medium at 2-8°C.